基于LC-MS/MS的刺蒺藜神经细胞保护作用物质基础研究
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摘要
目的:探讨刺蒺藜乙酸乙酯提取物对抗血管紧张素Ⅱ以及对人神经细胞的保护机制,通过分析有效成分、预测作用靶点,明确刺蒺藜神经保护的物质基础,以期寻找潜在的分子药物。方法:刺蒺藜乙酸乙酯层经过硅胶柱粗分制备,MTT法检测各部分抑制血管紧张素Ⅱ干预的人神经细胞系(SH-SY5Y)细胞活性的效果。液相色谱串联质谱(LC-MS/MS)检测效果最佳的刺蒺藜乙酸乙酯Fr.10部分,分析得到的具体成分。用药物吸收的主要原理来鉴定可吸收成分,预测有效成分的作用靶点。使用Gene Ontology分析、KEGG生物学通路对潜在靶点进行功能分析。结果:经过硅胶柱粗分得到的18个部分刺蒺藜乙酸乙酯提取物,经MTT法筛选显示刺蒺藜乙酸乙酯Fr.10效果最佳。LC-MS/MS共鉴定出708种化学成分,包括有机酸类、黄酮类、氨基酸类、酚胺类以及生物碱类等。质谱峰峰面积积分>6的化合物有18种。其中,甲基丙二酸、L-亮氨酸等11种成分可被吸收。这些潜在活性成分共调节551个靶点,作用主要集中在L-脯氨酸生物合成过程、线粒体呼吸链复合物Ⅱ、琥珀酸脱氢酶活性。涉及通路主要有柠檬酸循环、精氨酸和脯氨酸代谢等。结论:刺蒺藜乙酸乙酯提取物可通过多种途径发挥神经保护作用,对调节神经细胞的代谢和供能起到关键作用。
Objective: To explore the protective mechanism of the ethyl acetate extract of Tribulus terrestris Linn. against angiotensin Ⅱ and human neural cells. By analyzing the active ingredients and predicting the target of action, the material basis of the neuroprotection of Tribulus terrestris Linn. was clarified in order to find potential molecular drugs. Methods: The ethyl acetate layer of Tribulus terrestris Linn. was prepared by crude fractionation of silica gel column. The proliferation of human neural cell line(SHSY5 Y) inhibited by angiotensin Ⅱ was detected by MTT assay. Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to detect the best ethyl acetate Fr.10 part of the Tribulus terrestris Linn., and the specific ingredients were analyzed. The main principles of drug absorption are used to identify the absorbable ingredients and predict the target of the effective ingredients.Gene Ontology analysis and KEGG biological pathway were used for functional analysis of potential targets. Results: The 18 parts of the extract of Tribulus terrestris Linn. obtained by crude fractionation of silica gel column was screened by MTT method showed that the effect of ethyl acetate Fr.10 was the best. A total of 708 chemical components were identified by LC-MS/MS, including organic acids, flavonoids, amino acids, phenol amines, alkaloids and so on. There are 18 compounds with a mass spectral peak area integral greater than 6. Among them, eleven components such as methylmalonic acid and L-leucine can be absorbed. These potential active ingredients coordinate a total of 551 targets, mainly in the L-proline biosynthesis process, mitochondrial respiratory chain complexⅡ, and succinate dehydrogenase activity. The pathways involved include citrate circulation, arginine and proline metabolism.Conclusion: The ethyl acetate extract of Tribulus terrestris Linn. can exert neuroprotective effects through various pathways, which plays a key role in regulating the metabolism and energy supply of nerve cells.
Objective: To explore the protective mechanism of the ethyl acetate extract of Tribulus terrestris Linn. against angiotensin Ⅱ and human neural cells. By analyzing the active ingredients and predicting the target of action, the material basis of the neuroprotection of Tribulus terrestris Linn. was clarified in order to find potential molecular drugs. Methods: The ethyl acetate layer of Tribulus terrestris Linn. was prepared by crude fractionation of silica gel column. The proliferation of human neural cell line(SHSY5 Y) inhibited by angiotensin Ⅱ was detected by MTT assay. Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to detect the best ethyl acetate Fr.10 part of the Tribulus terrestris Linn., and the specific ingredients were analyzed. The main principles of drug absorption are used to identify the absorbable ingredients and predict the target of the effective ingredients.Gene Ontology analysis and KEGG biological pathway were used for functional analysis of potential targets. Results: The 18 parts of the extract of Tribulus terrestris Linn. obtained by crude fractionation of silica gel column was screened by MTT method showed that the effect of ethyl acetate Fr.10 was the best. A total of 708 chemical components were identified by LC-MS/MS, including organic acids, flavonoids, amino acids, phenol amines, alkaloids and so on. There are 18 compounds with a mass spectral peak area integral greater than 6. Among them, eleven components such as methylmalonic acid and L-leucine can be absorbed. These potential active ingredients coordinate a total of 551 targets, mainly in the L-proline biosynthesis process, mitochondrial respiratory chain complexⅡ, and succinate dehydrogenase activity. The pathways involved include citrate circulation, arginine and proline metabolism.Conclusion: The ethyl acetate extract of Tribulus terrestris Linn. can exert neuroprotective effects through various pathways, which plays a key role in regulating the metabolism and energy supply of nerve cells.
引文
[1]戴海龙,光雪峰,肖志成.高血压与认知功能障碍的研究进展.中华高血压杂志,2015,23(12):1135-1140
[2]李晨晔,马兰,李静,等.血管紧张素转化酶抑制剂对短暂性脑缺血发作合并高血压病患者认知功能的影响.哈尔滨医科大学学报,2016,50(1):54-58
[3]郭金昊,姜月华,杨传华,等.刺蒺藜对老年自发性高血压大鼠胸主动脉血管重塑的影响.中医杂志,2016,57(11):957-961
[4]翟凤国,周福波,李厚忠,等.蒺藜皂苷对局灶性脑缺血大鼠炎症反应和血脑屏障通透性遏影响.医药导报,2015,34(9):1131-1133
[5]赵外荣,施雯婷,郁丘婷,等.蒺藜化学成分分析及其皂苷类成分对心血管疾病作用的实验研究进展.上海中医药大学学报,2018,32(4):105-108
[6]Phillips O A,Mathew K T,Oriowo M A.Antihypertensive and vasodilator effects of methanolic and aqueous extracts of Tribulusterrestris in rats.J Ethnopharmacol,2006,104(3):351-355
[7]Li M,Guan Y,Liu J,et al.Cellular and molecular mechanisms in vascular smooth muscle cells by which total saponinextracted from Tribulus terrestris protects against artheros-clerosis.Cell Physiol Biochem,2013,32(5):1299-1308
[8]Zhu W,Du Y,Meng H,et al.A review of traditional pharmacological uses,phytochemistry,and pharmacological activities of Tribulus terrestris.Chem Cent J,2017,11(1):60
[9]Reshma P L,Sainu N S,Mathew A K,et al.Mitochondrial dysfunction in H9c2 cells during ischemia and amelioration with Tribulus terrestris L.Life Sci,2016,152:220-230
[10]Reshma P L,Lekshmi V S,Sankar V,et al.Tribulus terrestris(Linn.)attenuates gellular alte-rations induced by Ischemia in H9c2 cells via antioxidant Potential.Phytother Res,2015,29(6):933-943
[11]Berkman Z,Tanriover G,Acar G,et al.Changes in the brain cortex of rabbits on a cholesterol-rich diet following supplementation with a herbal extract of Tribulus terrestris.Histol Histopathol,2009,24(6):683-692
[12]Tuncer M A,Yaymaci B,Sati L,et al.Influence of Tribulus terrestris extract on lipid profile and endothelial structure in developingatherosclerotic lesions in the aorta of rabbit-son a highcholesterol diet.Acta Histochem,2009,111(6):488-500
[13]Gauthaman K,Adaikan P G.Effect of Tribulus terrestris on nicotinamide adenine dinucleotide phosphate-diaphorase activity and androgen receptors in rat brain.J Ethnopharmacol,2005,96(1-2):127-132
[14]Gabbi P,Ribeiro L R,JessiéMartins G,et al.Methylmalonate induces inflammatory and apo-ptotic potential:A link to glial activation and neurological dysfunction.J Neuropathol Exp Neurol,2017,76(3):160-178
[15]Sun A L,Ni Y H,Li XB,et al.Urinary methylmalonic acid as an indicator of early vita-min B12 deficiency and its role in polyneuropathy in type2 diabetes.JDiabetesRes,2014,2014:921616
[16]Viegas C M,Zanatta?,Grings M,et al.Disruption of redox homeostasis and brain damage caused in vivo by methylmalonic acid and ammonia in cerebral cortex and striatum of developing rats.Free Radic Res,2014,48(6):659-669
[17]Morris G,Anderson G,Dean O,et al.The glutathione system:a new drug target in neu-roimmune disorders.Mol Neurobiol,2014,50(3):1059-1084
[18]Tsverava L,Lordkipanidze T,Lepsveridze E,et al.Myoinositol attenuates the cell loss and biochemical changes induced by kainic acid status epilepticus.Biomed Res Int,2016,2016:2794096
[19]Matsuo K,Yabuki Y,Fukunaga K.Combined lcitrulline and glutathione administration prevents neuronal cell death followingtransient brain ischemia.BrainRes,2017,1663:123-131
[20]Lopes-Azevedo S,Busnardo C,Corrêa FMA.Central mechanism of the cardiovascular responses caused by L-proline microinjected into the paraventricular nucleus of the hypothalamus in unanesthetized rats.Brain Res,2016,1652:43-52
[21]Jodeiri Farshbaf M,Kiani-Esfahani A.Succinate dehydrogenase:Prospect for neurodegener-ative diseases.Mitochondrion,2018,42:77-83
[22]Cai S,Ling C,Lu J,et al.EGAR,A Food Protein-Derived Tetrapeptide,Reduces Seizure Activity in PentylenetetrazoleInduced Epilepsy Models Throughα-Amino-3-Hydroxy-5-Me-thyl-4-Isoxazole Propionate Receptors.Neurotherapeutics,2017,14(1)212-226
[23]Sha D,Wang L,Zhang J,et al.Cocaine-and amphetamineregulated transcript peptide increases mitochondrial respiratory chain complexⅡactivity and protects against oxygen-glucose deprivation in neurons.Brain Res,2014,25:107-113
[24]Westergaard N,Waagepetersen H S,Belhage B,et al.Citrate,a ubiquitous key metabolite with regulatory Function in the CNS.Neurochem Res,2017,42(6):1583-1588
[25]Patin F,Corcia P,Vourc’h P,et al.Omics to explore amyotrophic lateral sclerosis evolution:the central role of arginine and proline metabolism.Mol Neurobiol,2017,54(7):5361-5374
[26]Engskog M K,Ersson L,Hagl?f J,et al.β-N-Methylamino-Lalanine(BMAA)perturbs alanine,aspartate and glutamate metabolismpathways in human neuroblastoma cells as determined by metabolic profiling.Amino Acids,2017,49(5):905-919
[2]李晨晔,马兰,李静,等.血管紧张素转化酶抑制剂对短暂性脑缺血发作合并高血压病患者认知功能的影响.哈尔滨医科大学学报,2016,50(1):54-58
[3]郭金昊,姜月华,杨传华,等.刺蒺藜对老年自发性高血压大鼠胸主动脉血管重塑的影响.中医杂志,2016,57(11):957-961
[4]翟凤国,周福波,李厚忠,等.蒺藜皂苷对局灶性脑缺血大鼠炎症反应和血脑屏障通透性遏影响.医药导报,2015,34(9):1131-1133
[5]赵外荣,施雯婷,郁丘婷,等.蒺藜化学成分分析及其皂苷类成分对心血管疾病作用的实验研究进展.上海中医药大学学报,2018,32(4):105-108
[6]Phillips O A,Mathew K T,Oriowo M A.Antihypertensive and vasodilator effects of methanolic and aqueous extracts of Tribulusterrestris in rats.J Ethnopharmacol,2006,104(3):351-355
[7]Li M,Guan Y,Liu J,et al.Cellular and molecular mechanisms in vascular smooth muscle cells by which total saponinextracted from Tribulus terrestris protects against artheros-clerosis.Cell Physiol Biochem,2013,32(5):1299-1308
[8]Zhu W,Du Y,Meng H,et al.A review of traditional pharmacological uses,phytochemistry,and pharmacological activities of Tribulus terrestris.Chem Cent J,2017,11(1):60
[9]Reshma P L,Sainu N S,Mathew A K,et al.Mitochondrial dysfunction in H9c2 cells during ischemia and amelioration with Tribulus terrestris L.Life Sci,2016,152:220-230
[10]Reshma P L,Lekshmi V S,Sankar V,et al.Tribulus terrestris(Linn.)attenuates gellular alte-rations induced by Ischemia in H9c2 cells via antioxidant Potential.Phytother Res,2015,29(6):933-943
[11]Berkman Z,Tanriover G,Acar G,et al.Changes in the brain cortex of rabbits on a cholesterol-rich diet following supplementation with a herbal extract of Tribulus terrestris.Histol Histopathol,2009,24(6):683-692
[12]Tuncer M A,Yaymaci B,Sati L,et al.Influence of Tribulus terrestris extract on lipid profile and endothelial structure in developingatherosclerotic lesions in the aorta of rabbit-son a highcholesterol diet.Acta Histochem,2009,111(6):488-500
[13]Gauthaman K,Adaikan P G.Effect of Tribulus terrestris on nicotinamide adenine dinucleotide phosphate-diaphorase activity and androgen receptors in rat brain.J Ethnopharmacol,2005,96(1-2):127-132
[14]Gabbi P,Ribeiro L R,JessiéMartins G,et al.Methylmalonate induces inflammatory and apo-ptotic potential:A link to glial activation and neurological dysfunction.J Neuropathol Exp Neurol,2017,76(3):160-178
[15]Sun A L,Ni Y H,Li XB,et al.Urinary methylmalonic acid as an indicator of early vita-min B12 deficiency and its role in polyneuropathy in type2 diabetes.JDiabetesRes,2014,2014:921616
[16]Viegas C M,Zanatta?,Grings M,et al.Disruption of redox homeostasis and brain damage caused in vivo by methylmalonic acid and ammonia in cerebral cortex and striatum of developing rats.Free Radic Res,2014,48(6):659-669
[17]Morris G,Anderson G,Dean O,et al.The glutathione system:a new drug target in neu-roimmune disorders.Mol Neurobiol,2014,50(3):1059-1084
[18]Tsverava L,Lordkipanidze T,Lepsveridze E,et al.Myoinositol attenuates the cell loss and biochemical changes induced by kainic acid status epilepticus.Biomed Res Int,2016,2016:2794096
[19]Matsuo K,Yabuki Y,Fukunaga K.Combined lcitrulline and glutathione administration prevents neuronal cell death followingtransient brain ischemia.BrainRes,2017,1663:123-131
[20]Lopes-Azevedo S,Busnardo C,Corrêa FMA.Central mechanism of the cardiovascular responses caused by L-proline microinjected into the paraventricular nucleus of the hypothalamus in unanesthetized rats.Brain Res,2016,1652:43-52
[21]Jodeiri Farshbaf M,Kiani-Esfahani A.Succinate dehydrogenase:Prospect for neurodegener-ative diseases.Mitochondrion,2018,42:77-83
[22]Cai S,Ling C,Lu J,et al.EGAR,A Food Protein-Derived Tetrapeptide,Reduces Seizure Activity in PentylenetetrazoleInduced Epilepsy Models Throughα-Amino-3-Hydroxy-5-Me-thyl-4-Isoxazole Propionate Receptors.Neurotherapeutics,2017,14(1)212-226
[23]Sha D,Wang L,Zhang J,et al.Cocaine-and amphetamineregulated transcript peptide increases mitochondrial respiratory chain complexⅡactivity and protects against oxygen-glucose deprivation in neurons.Brain Res,2014,25:107-113
[24]Westergaard N,Waagepetersen H S,Belhage B,et al.Citrate,a ubiquitous key metabolite with regulatory Function in the CNS.Neurochem Res,2017,42(6):1583-1588
[25]Patin F,Corcia P,Vourc’h P,et al.Omics to explore amyotrophic lateral sclerosis evolution:the central role of arginine and proline metabolism.Mol Neurobiol,2017,54(7):5361-5374
[26]Engskog M K,Ersson L,Hagl?f J,et al.β-N-Methylamino-Lalanine(BMAA)perturbs alanine,aspartate and glutamate metabolismpathways in human neuroblastoma cells as determined by metabolic profiling.Amino Acids,2017,49(5):905-919